A G protein is a heterotrimeric guanine nucleotide binding regulatory protein, generally localized to the inner surface of the plasma membrane where it transduces signals from transmembrane receptors to intracellular effectors. In the resting state, the G protein exists as a trimer containing .alpha., .beta., and .gamma. subunits. Interaction of the G protein with agonist-bound receptor stimulates the exchange of GTP for GDP on the a subunit, and the .alpha. and .gamma. subunit dissociate from the activated a subunit. The GTP-bound .alpha. subunit and the free .beta./.gamma. complex can act independently on downstream effectors to modulate second messenger concentrations. The hydrolysis of GTP to GDP deactivates the a subunit, thereby allowing the reassociation of the .alpha. and .beta./.gamma. complex the recoupling of the heterotrimer to the receptor (Casey, P. J., Curr. Opin. Cell Biol. 6: 219-225, 1994).
Platelets are small disc-shaped cell fragments which undergo a rapid transformation when they encounter sites of vascular damage. They become more spherical and extrude pseudopodia, their fibrinogen receptors are activated leading to aggregation, and they release their granule contents and eventually they form a plug which is responsible for primary hemostasis (Siess, W., Physiol. Rev. 69: 58-178, 1989). Activation of platelets is also implicated in the pathogenesis of unstable angina, myocardial infarction and stroke (Packham, M. A., Can J. Physiol Pharmacol. 72: 278-284).
Several physiological substances are involved in the activation of platelets such as collagen, which is exposed at the subendothelial surfaces, thrombin, generated by the coagulation cascade, and thromboxane A.sub.2 (TXA.sub.2) and ADP, which are released from activated platelets. Collagen binds to several platelet membrane proteins including integrin .alpha..sub.2 .beta..sub.1 leading to platelet activation through the release of TXA.sub.2 and ADP (Shattil, S. J., et al., Curr. Opin. Cell Biol. 6: 695-704, 1994). In contrast, thrombin, TXA.sub.2, and ADP, activate G-protein coupled receptors directly and induce platelet aggregation and granule release (Hourani, S. M, and Cusack, N. J., Pharmacol. Rev. 43: 243-298, 1991). The receptors for these platelet activators have been shown to couple to several G-proteins, including G.sub.i, G.sub.q, G.sub.12, and G.sub.13 (see, for example, Shenker, A., et al., J. Biol. Chem. 266: 9309-9313, 1991). These G-proteins have been shown to regulate different effectors (Neer, E. I., Cell 80: 246-257, 1995; Hepler, J. R., and Gilman, A. G., Trends Biochem. Sci. 17: 383-387, 1992).
The major events involved in platelet activation are believed to be the result of the activation of .beta.-isoforms of phospholipase C (PLC) leading to the generation of inositol 1,4,5 triphosphate and diacylglycerol. It is not clear whether G-protein mediated release of ".beta.".gamma.-subunits or the activation of G.sub.q is the predominant mechanism for PLC-.beta. activation in platelets. Platelets mainly contain two isoforms, PLC-.beta.2 and PLC-.beta.3 (Lee, S. B., et al., Blood 88: 1684-1691, 1996; Bonno, Y., et al., J. Biol. Chem. 271: 14989-14994, 1996). While PLC-.beta.2 is only weakly activated by G.alpha..sub.q/11 when compared with PLC-.beta.3, both enzymes appear to be regulated by G-protein .beta..gamma.-subunits. Most mammalian tissues express G.alpha..sub.q together with its close structural and functional homolog G.alpha..sub.11 Platelets are, however, an exception.